Dioxetane compounds for the chemiluminescent detection of proteases, methods of use and kits therefore

ABSTRACT

1,2-dioxetane compounds bearing a proteolytic enzyme specific amino acid or peptide are provided, which amino acid or peptide can be removed by action of the corresponding protease. When the amino acid or peptide is removed, the 1,2-dioxetane decomposes with chemiluminescence, the generation of light providing a rapid, ultra-sensitive and convenient means for detecting the presence of the protease in the sample being inspected. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of protease present. Immunoassays, as well as DNA hybridization and DNA probe assays are provided.

This is a continuation of application Ser. No. 08/728,990 filed on Oct.11, 1996, now U.S. Pat. No. 5,843,681 which is a continuation of Ser.No. 08/385,788, filed on Feb. 9, 1995, allowed, now U.S. Pat. No.5,591,591.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention pertains to chemiluminescent compounds, their use inassays for the detection of proteases, and kits comprising the compoundsand other elements used in protease detection assays. Specifically,dioxetane compounds bearing a proteolytic enzyme-specific amino acid orpeptide which, when removed by enzymatic reaction, causes the dioxetaneto decompose and chemiluminesce, is provided. These compounds, whenadded to a sample suspected of containing the protease, provide a rapid,ultrasensitive and convenient means for detecting the presence of theprotease in the sample. The amount of light generated, or degree ofchemiluminescence, can be correlated with the amount of proteasepresent.

BACKGROUND OF THE INVENTION

Applications naming one or more of the inventors herein, as inventors,and assigned to Tropix, have clearly established 1,2-dioxetanes aschemiluminescence compounds which can be used as reporters inultrasensitive assays that can be conducted quickly, without resort toexotic conditions or elaborate apparatus, for the detection of a varietyof biological materials. Among these are U.S. Pat. Nos. 4,931,223;4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772;5,220,005; 5,225,584; 5,326,882; 5,330,900 and 5,336,596. Other patentscommonly assigned with this application have issued, and still otherapplications are pending. Together, this wealth of patent literatureaddresses 1,2-dioxetanes, stabilized by a typically polycyclic group,preferably spiroadamantane bonded to one of the carbons of the dioxetanering, and a moiety bonded to the remaining carbon of the dioxetane ringwhich is electron sensitive, such that deprotection of the electronsensitive moiety, typically an aryl group, more preferably a phenyl ornaphthyl group, leads to an anion, generally an oxyanion, which isunstable, and decomposes. Through decomposition, the O--O bond isbroken, and a photon is generated. The same carbon atom to which thiselectron sensitive moiety is bonded may bear an alkoxy or otherelectron-active group. Methoxy is a preferred moiety.

The first of the dioxetanes of this class commercialized was3-(4-methoxy-spiro 1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7!decan!-4-yl)phenyl phosphate, particularly the disodium salt, generallyknown as AMPPD. This compound has been commercialized by Assignee ofthis application, Tropix, Inc., as well as a company of Detroit, Mich.,Lumigen, Inc. The primary inventor of Lumigen, A. Paul Schaap, has beengranted several patents on related technology.

Superior performance can be obtained by selective substitution on thespiroadamantane ring. Substitution, at either bridgehead carbon, with anelectron-active species, such as chlorine, improves reaction speed andsignal-to-noise ratio (S/N). The chlorine-substituted counterpart ofAMPPD, CSPD, has been widely commercialized by Tropix, Inc. of Bedford,Mass. "Third-generation" dioxetane compounds of similar structure,wherein the phenyl or naphthyl moiety also bears an electron-activesubstituent, such as chlorine, offer further improvements inperformance, and have been commercialized by Tropix, Inc. The phosphatemoieties is available under the trademarks CDP and CDP-Star.

A common characteristic of all these dioxetanes is the blocking ormasking group on the phenyl or naphthyl moiety. Groups which areenzyme-specific substrates are employed, such that, when admixed withthe enzyme, the blocking group is removed by the enzyme, leaving anelectron rich oxygen moiety attached to the phenyl or naphthylsubstituent. Typically, this blocking group has been a phosphate,although other blocking groups, such as a galactoside have also beenused. Representative blocking groups are set forth in the patents listedabove, which are incorporated herein by reference. These blocking groupshave been substrates for enzymes which are specific for the blockinggroup, the enzymes either being selected as enzymes of interest orpotential interest in a biological fluid, or non-endogenous enzymeswhich may be coupled to a particular target moiety of the sample, theirtriggering of the dioxetane to generate light being thus evidence of theanalyte in the sample to which the non-endogenous enzyme is coupled.Alkaline phosphatase has been the dominant enzyme of interest as atriggering agent.

The existing literature on dioxetanes does not describe a triggerabledioxetane, that is, a dioxetane which can be "deprotected" to inducedecomposition and chemiluminescence that can be used to detect, or betriggered by, proteolytic enzymes, that is, peptidases. These enzymesare involved in the life cycle of proteins. Further, proteolytic enzymesare also involved in the processing of proteins, hormones, receptors,growth factors, fertilization, activation of regulatory proteasesinvolved in blood coagulation, fibrinolysis and the complement reaction,and other cellular functions. Thus, the presence or absence of aparticular proteolytic enzyme in a biological sample may well indicatethe presence or absence of a particular disease state, pathogeniccondition or organic syndrome. Families of proteolytic enzymes includeserine proteases (chymotrypsin and subtilisin), cysteine proteases(papain), aspartic proteases (penicillopepsin) and metalloproteases(carboxypeptidase and thermolysin).

In addition to being of particular interest as organic moieties thatconstitute diagnostic markers, protease enzymes are also of considerableinterest as enzyme labels, and are used in detergent production andleather processing. Examples of diagnostic protease markers includecathepsin B (cancer), cathepsin G (emphysema, rheumatoid arthritis,inflammation), cathepsin L (cancer), elastase (emphysema, rheumatoidarthritis, inflammation), plasminogen activator (thrombosis, chronicinflammation, cancer), and urokinase (cancer). The use of alkalineproteases in detergents is expected to increase. Assays for proteasedetection are therefore needed to monitor protein stability in variousbiological and commercial processes.

Known protease assay conditions are listed in the following table:

    ______________________________________    Protease     Assay Conditions    ______________________________________    acylaminoacyl peptidase                 Ac--Ala-p-nitroanilide in Tris                 HCl, pH 7.5, 37° C.    aminopeptidase M                 L--Leu-p-nitroanilide in 60 mM Na                 phosphate buffer, pH 7-7.5, 37° C.    cathepsin B  Bz--Phe--Arg--NMec, pH 5.5--6.0, 37° C.    cathepsin G  MeO--Suc--Ala--Ala--Pro--Phe-p-                 nitroanilide, pH 7.5, 25° C.    cathepsin L  Cbz--Phe--Arg--NMec in 340 mM Na                 acetate, 60 mM acetic acid, 4 mM                 disodium EDTA, pH 5.5, 8 mM                 dithiothreitol, 30° C.    elastase     Suc--(Ala).sub.3 -p-nitroanilide, 0.05%                 Triton X-100 in plastic tubes,                 pH 7.8, 25° C.    subtilisin   denatured hemoglobin degradation, pH 7-8    plasminogen activator                 Val--Leu--Lys-p-nitroanilide in                 50 mM Tris--HCl, pH 8.5, 37° C.    urokinase    Val--Phe--Lys-p-nitroanilide in                 50 mM Tris--HCl, pH 8.5, 37° C.    ______________________________________

Some of the known and potential applications for proteolytic enzymes arelisted in the following table:

    ______________________________________    Protease    Applications    ______________________________________    Aminopeptidase                detection of gram-neg bacteria    Cathepsin B cancer marker    Cathepsin G emphysema, rheumatoid arthritis, inflammation                marker    Cathepsin L cancer marker    Elastase    emphysema, rheumatoid arthritis, inflammation                marker    Subtilisin  used in leather processing as a depilatory,                detergents, protein hydrolysate production (e.g.                Optimase and Opticlean enzymes by Solvay                Enzymes),    Plasminogen Activator                thrombosis, chronic inflammation, cancer marker    Urokinase   cancer marker    General     Protease screen, screening of protein formulations                for the presence of proteases.                Sensitive detection of DNA in solution and on                blots utilizing thermophilic enzymes such as                thermolysin which would enable the incorporation                of the enzyme label during the DNA preparation                procedure such as high temperature denaturation                and PCR amplification.    ______________________________________

See also generally Proteolytic Enzymes--A Practical Approach, IRL Press,pp. 233-40 (1989).

Known Protease Assay Methods

The basic methods of the detection of proteolytic enzymes consist of anenzymatic reaction with a suitable substrate and the detection of theproduct of this reaction. Thus, a known protease will cleave a syntheticpeptide which is a substrate for such enzyme. Synthetic substrates areamino acids and peptides. Specific substituents are added to the α-NH₂or α-COOH groups to either block the amino or the carboxyl to produce asubstrate which can be recognized and cleaved by a specific protease.Additionally, the added group may add a "signaling" property to asubstrate such that the products of the substrate-enzyme reaction can bedetected and monitored by absorption or fluorescence spectrometry.

There are several commercially available assays for proteolytic enzymes.Promega offers a PepTag™ Protease Assay. This is a qualitative assaywhich detects the proteolysis of small dye-linked peptides. Since thedigestion of these peptides changes their molecular weight and charge,they can be detected using agarose gel electrophoresis. This assay,however, is cumbersome, not sensitive and not quantitative. There arealso several fluorogenic substrates for proteolytic enzymes availablefrom Sigma, Calbiochem and Molecular Probes.

Accordingly, it remains an object of those of skill in the art toprovide a quick, reliable and sensitive assay for the detection ofproteolytic enzymes, either as markers, commercial indicators, orlabels, that can be both qualitative and quantitative. It is a furtherobjection of those of skill in the art to provide dioxetane compoundswhich can be triggered by proteolytic enzymes to decompose andchemiluminesce, in a fashion analogous to those reflected in theliterature, which undergo decomposition from the oxyanion moiety.

SUMMARY OF THE INVENTION

1,2-dioxetanes of the following general formula I can be used to detectthe presence of proteolytic enzymes in a sample. ##STR1## Wherein X¹, X²and Y are independently hydrogen or an electron active substituent,wherein R is an alkyl, straight-chain or branched chain of 1-20 carbonatoms or cycloalkyl or polycycloalkyl of 3-20 carbon atoms, and whereinZ is an amino acid, or a polypeptide, wherein Z is an enzyme-specificsubstrate. These dioxetanes are stable under storage conditions, andsufficiently soluble in water or an aqueous preparation so as to serveas a chemiluminescent reporter molecule for detection of proteolyticenzymes which cleave the enzyme-specific substrate Z. When the Z groupis removed, the arylamine decomposes, breaking the O--O bond of thedioxetane ring and generating chemiluminescence as in the case ofconventional 1,2-dioxetanes triggered through the formation of theoxyanion addressed in the literature. To improve solubility, any one ormore of X¹, X² or Y can be selected to be a solubilizing agent, that is,an agent which improves hydrogen-bonding, so as to improve thesolubility of the dioxetane reporter molecule in the sample, which isgenerally aqueous in nature, particularly where biological assays arebeing conducted. For aqueous solubility, R is preferably 6 carbon atomsor less. Certain commercial processes may require organic preparationsand organic solvents. In these cases, R may be of greater size, and X¹,X² and Y selected so as to improve solubility in the selected solvent.

Z in the above formula is selected to be a proteolytic enzyme-specificsubstrate. By this, it is intended that Z be particularly formulated tobe a substrate which is cleaved by a specific proteolytic enzyme, orclass of enzymes, such that other enzymes likely to be in the sampleassayed will not cleave the Z moiety, and thus, any chemiluminescencedetected will be traceable directly to the presence of the enzymeitself. The invention is not limited to any specific amino acid orpeptide sequence, and is open to both commercially available peptidesequences and groups to be developed for specific assays. It should benoted that certain proteolytic assays are specific for a single aminoacid. In this situation, Z can be confined to a single amino acid. Oncethe peptide or amino acid is removed, the resulting dioxetane amine willdecompose with chemiluminescence, just as in the case of the oxyanionsof the existing dioxetane literature. The amount of chemiluminescencecan be correlated with the amount of enzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemiluminescence trace obtained by subtilisin triggering of1,2-dioxetane subtilisin-specific substrate.

FIG. 2. A graph reflecting the chemiluminescent signal obtained bytriggering 1,2-dioxetane substrate for subtilisin with serial dilutionsof subtilisin at 37° C. FIG. 2A is presented in light units, while FIG.2B provides a measure of the signal-to-noise (S/N) ratio.

FIG. 3. FIG. 3 provides information on the experiment reflected in FIG.2, with further incubation for one hour at 45° C.

DETAILED DESCRIPTION OF THE INVENTION

The dioxetanes claimed herein begin with starting materials akin tothose employed for the phenoxy-substituted dioxetanes of the literature.Thus, the chemical structure of the dioxetane compounds claimed hereinis analogous to that of the phenoxydioxetanes of the prior art, save forthe fact that the oxygen moiety linked to the phenyl group substitutedon the dioxetane, or naphthyl group substituted on the dioxetane, isreplaced with an amino group. The compounds have the formula ##STR2##wherein X¹, X² and Y are independently H or an electron active (electronwithdrawing or electron donating) moiety. If the group is an electronactive group, it may also be selected to improve the solubility of thedioxetane in the solvent or medium selected. Most commonly, the sampleto be assayed is prepared in an aqueous medium, and accordingly, thesolubilizing moiety or moieties will be those that facilitate hydrogenbonding. Depending whether X¹, X² and Y are selected as hydrogen orelectron active, the identity may affect, in addition to solubility,half-life (T1/2) of the decomposition reaction, chemiluminescent yield,and signal to noise ratio (S/N). Suitable substituents and their impacton the dioxetane chemiluminescence, are discussed in connection withphenoxy-substituted dioxetanes and disclosed and claimed in U.S. Pat.No. 5,330,900. Thus, each of the substituents at the bridge-head carbonson the adamantane ring may individually represent, in addition tohydrogen, a hydroxyl group, a halosubstituent, a hydroxy lower alkylgroup, a halo lower alkyl group, a phenyl group, a halo phenyl group, analkoxy phenyl group, a hydroxy alkoxy group, a cyano group, or an amidegroup, Similarly, the identity of Y may be hydrogen or election active.If selected without regard to solubility characteristics, exemplaryidentities for Y include Cl, OM, OAr, NM₃ +, NHCOM, NMCOM¹, NHCOAr,NHCOOAr, NHCOOM, NMCOOM¹, CM₃, NO₂, COOM, COOAr, NHSO₂ OM, NHSO₂ AR,CF₃, Ar, M, SiM₃, SiAr₃, SiArM₂, SO₂, NHCOM, SO₂ NHCOAr, SO₂ M, SO₂ AR,SM and SAr wherein M and M¹ are independently C1-6 alkyl, and Ar isphenyl or naphthyl. A preferred substituent is chlorine, methoxy, alkylor amido. Compounds of this type, bearing a phenoxy, rather than aphenyl amine substituent are disclosed and claimed in pending U.S.patent application Ser. No. 08/057,903 now U.S. Pat. No. 5,538,847. Thesame is incorporated herein by reference.

The point of substitution of Y and the NH-Z moiety on the aryl ring mayinfluence the chemiluminescence obtained and the T_(1/2) of theresulting dioxetane. Where the dioxetane bears a phenyl substituent, thepreferred point of substitution for NH-Z is meta with respect to thepoint of attachment to the dioxetane, and where substituting on anaphthyl moiety, the NH-Z moiety should be at a position disjoint fromthe point of attachment to the dioxetane moiety. Preferred positions forY when not hydrogen, are positions other than ortho with respect to thepoint of attachment to the dioxetane.

The identity of moiety Z is not specifically limited, save that it mustbe an enzyme-cleavable group, such that when admixed with a samplecontaining the enzyme of interest, the enzyme cleaves the moiety Z,causing the dioxetane amine to undergo spontaneous decomposition,generating light. A large number of substrates for proteolytic enzymessuitable as candidates for moiety Z are commercially available, andothers have been identified. Z is typically an amino acid or peptide,although some proteases recognize only amino acids or peptides withblocking groups, such as N-carbobenzoxy (N-Cbz or N-Z), N-succinyl(N-suc), N-methoxysuccinyl (N-MeOsuc), N-acyl (N-Ac) or N-benzoyl(N-Bz). Z is an amino acid, peptide, or the same protected with ablocking group, such that the protease recognizes Z as a cleavage site.The following identities for moiety Z are exemplary only, and are notintended to limit the scope of the invention.

    ______________________________________    Protease         (Amino Acid or Peptide Z--)    ______________________________________    Alzheimer's Disease                     Cbz--Val--Lys--Met--    (Amyloid A4-generating enzymes)    Aminopeptidase   Arg--                     Ala--                     Ala--Ala--Phe--                     Glu--                     Gly--Pro--Leu--                     Gly--Pro--Met--                     Leu--    Cathepsin B      Arg--                     Cbz--Arg--Arg--                     Cbz--Phe--Arg--                     Bz--Arg--                     Suc--Ala--Ala--Pro--Phe                     Cbz--Ala--Arg--Arg--    Cathepsin C      Gly--Phe--                     Ala--Arg--                     Bz--Arg--Gly--Leu--                     Gly--Arg--    Cathepsin D      Bz--Arg--Gly--Phe--Phe--Pro--    Cathepsin G      Suc--Ala--Ala--Phe--                     Suc--Phe--Leu--Phe--                     Suc--Val--Pro--Phe--                     MeOSuc--Ala--Ala--Pro--Met--    Cathepsin H      Arg--                     Leu--    Cathepsin L      Cbz--Phe--Arg--    Elastase         Ac--Ala--Ala--Ala                     Suc--Ala--Ala--Ala--                     Boc--Ala--Ala--Ala--                     Suc--Ala--Ala--Val--                     Suc--Ala--Pro--Ala--                     Ac--Ala--Pro--Ala--                     Suc--Ala--Ala--Pro--                     Ac--Ala--Ala--Pro--Ala--                     Boc--Ala--Ala--Pro--Ala--                     Glu--Ala--Ala--Pro--Leu--                     Suc--Ala--Ala--Pro--Met--                     MeOSuc--Ala--Ala--Pro--Val--                     Boc--Ala--Ala--Pro--Val--                     Suc--Leu--Leu--Val--Tyr--                     Pro--Phe--Arg--                     Cbz--Gly--    Endopeptidase    Cbz--Gly--Gly--Leu--    Subtilisin       Cbz--Ala--Ala--Leu--                     Boc--Gly--Gly--Leu--                     Cbz--Gly--Gly--Leu    Thermolysin      Abz--Ala--Ala--Phe--Phe--                     Cbz--Phe--Arg--                     Cbz--Phe--Pro--Arg--    tPA              Cbz--Gly--Gly--Arg--                     Bz--Val--Gly--Arg--                     Boc--Val--Gly--Arg--    Urokinase        Cbz--Gly--Gly--Arg--                     Glt--Gly--Arg--                     Gly--Arg--                     Cbz--Val--Gly--Arg--    ______________________________________     Key:     Abz 2aminobenzoyl     Ac acetyl     Bz benzoyl     Glt glutaryl     Suc succinyl     Cbz carbobenzoxy     pGlu pyroglutamyl     Abu 2aminobutyric acid     pyr pyroglutamic acid

There are many commercially available peptides which are substrates forproteolytic enzymes and can be used to append suitable 1,2-dioxetanes togenerate chemiluminescent substrates for such enzymes. The list ofcommercial protease susbtrates is shown in the following table:

    ______________________________________    Protease       Substrates    ______________________________________    Aminopeptidase Leu--X                   Ala--X                   Arg--X                   Glu--X    Cathepsin B    Arg--X                   Cbz--Arg--Arg--X                   Cbz--Phe--Arg--X                   Cbz--Ala--Arg--Arg--X    Cathepsin H    Arg--X                   Leu--X    Cathepsin G    Suc--Phe--Leu--Phe--X                   Suc--Val--Pro--Phe--X                   Suc--Ala--Ala--Phe--X                   Suc--Phe--Pro--Phe--X    Cathepsin L    Cbz--Phe--Arg--X    Elastase       Ac--Ala--Ala--Ala--X                   Suc--Ala--Ala--Val--X                   Suc--Ala--Ala--Pro--X                   Suc--Ala--Pro--Ala--X                   MeOSuc--Ala--Ala--Pro--Val--X    Subtilisin     Cbz--Ala--Ala--Leu--X                   Cbz--G1y--Gly--Leu--X    Urokinase      Cbz--Gly--Gly--Arg--X                   Cbz--Val--Gly--Arg--X    ______________________________________     X = pnitroanilide, 7amido-4-methylcoumarin, or     7amido-4-trifluoromethylcoumarin

The specificity of a substrate is dictated by the enzyme's recognitionof specific peptide sequences. The specific cleavage sites forproteolytic enzymes are listed in the following table:

    ______________________________________    Protease     Specificity   Preferred Sites    ______________________________________    aminopeptidase N                 nonspecific   Ala, Arg, Glu, Leu    cathepsin B  basic amino acid                               Lys, Arg    cathepsin G  aromatic amino acid                               Phe, Tyr    cathepsin L  Arg           Arg    elastase     uncharged     Ala, Val                 nonaromatic    subtilisin   nonspecific   Leu    plasminogen activator                 capped        Lys, Arg                 basic amino acid    urokinase    basic amino acid                               Lys, Arg    ______________________________________

Syntheses of Protease Dioxetane Substrates

The following exmaples are representative syntheses of dioxetaneprotease substrates and should not limit the scope of the claims. N-Cbzand N-Ac are abbreviations for N-carbobenzoxy and N-acetyl respectively.

    ______________________________________    Dioxetane            Peptide chain   Substrate for    ______________________________________     3      Leu--Gly--Gly--N--Cbz                            subtilisin     9      Arg             cathepsins B/H                            aminopeptidase    10      Arg--Ala--N--Cbz                            trypsin    11      Arg--Gly--Gly--N--Cbz                            tissue plasminogen activator    12      Arg--Gly--Val--N--Cbz                            urokinase    17      Ala--Ala--N--Cbz                            elastase    18      (Ala).sub.3 --N--Ac                            elastase    19      (Ala).sub.4 --N--Cbz                            elastase    ______________________________________

3-(Methoxytricyclo 3.33.1.1³,7!dec-2-ylidenemethyl)-1-anilinyl-Leu-Gly-Gly-N-Cbz(2). Enol etheraniline 1 (206 mg, 0.77 mmol, synthesized as described in SBIR Grant #1R43 HG00196-01) was dissolved in 4 ml anhydrous dimethylformamide (DMF)and 4 ml anhydrous pyridine (py) under argon. N-Cbz-Gly-Gly-Leu (1.2 g,3.2 mmol), 1- 3-(dimethylamino)propyl!-3-ethylcarbodiimide HCl (606 mg,3.2 mmol) and 1-hydroxybenzotriazole (53 mg, 0.4 mmol) were added underargon at room temperature. The reaction was stirred for 1 hr,partitioned between ethyl acetate and water, and the organic layer waswashed successively with dilute brine and dilute sodium bicarbonatesolutions. The organic layer was dried over NA₂ SO₄, evaporated andpurified on a silica gel column (30-100% EtOAc/hexanes) to yield 367 mg(76%) of enol ether peptide 2 as a white foam.

¹ H NMR (CDCI₃, ppm): 0.82 (3H, d, J=6 Hz); 0.86 (3H, d, J=6 Hz);1.58-1.99 (15H, m); 2.56 (1H, br s); 3.16 (1H, br s); 3.20 (3H, s); 3.88(1H, m); 3.91 (2H, m); 3.97 (2H, m); 4.65 (1H, br s); 5.03 (2H, s); 5.87(1H, br s); 6.97 (1H, d, J=7.5 Hz); 7.16 (1H, t, J=8 Hz); 7.24 (5H, 6);7.43 (1H, br s); 7.49 (2H, m); 8.92 (1H, br s)

3-(4-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-Leu-Gly-Gly-N-Cbz (3). A solution of enol etherpeptide 2 (212.6 mg, 0.34 mmol) and 5,10,15,20-tetraphenyl-2-1H,23H-porphine (TPP, 0.6 ml of a 2% solution in CHCl₃ by weight) in 10ml chloroform was irradiated with a 250W, high pressure sodium lamp at10° C. while passing a stream of oxygen through the solution. A 5-milpiece of Kapton polyimide film (DuPont) placed between the lamp and thereaction mixture filtered out unwanted UV radiation. Analytical tlc andhplc showed complete dioxetane formation upon irradiating 8 min. Thesolvent was removed under vacuum at 0° C. and pumped dry. The residuallight yellow foam was dissolved in 1 ml acetonitrile (hplc grade) andstored overnight in the freezer to allow crystal formation. Theacetonitrile was pipeted off the white precipitate, the precipitate waswashed with 1 ml acetonitrile and then dried on high vacuum to give 152mg (77%) of white crystalline dioxetane 3.

¹ H NMR (DMSO-d₆, ppm): 0.87 (3H, d, J=6.35 Hz); 0.89 (3H, d, J=6.35Hz); 0.92 (1H, d); 1.19 (1H, d, J=10.8 Hz); 1.44-1.75 (13H, m); 2.07(1H, br s); 2.90 (1H, br 6); 3.10 (3H, s); 3.64 (2H, d, J=6 Hz); 3.76(2H, d, J=6Hz); 4.44 (1H, m); 5.01 (2H, s); 7.30 (1H, m); 7.33 (5H, m);7.41 (1H, t, J=7.8 Hz); 7.54 (1H, t, J=6 Hz); 7.74 (1H, d, J=7.8 Hz);7.88 (1H, br s); 8.10 (1H, d, J=7.8 Hz); 8.17 (1H, m).

3-(Methoxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)-1-anilinyl-N.sup.α,N^(G), N^(G1) -tri-Cbz-L-Arg (4). Enol ether aniline 1 (31 mg, 0.115mmol) was dissolved in 0.5 ml anhydrous dimethylformamide and 0.5 mlanhydrous pyridine under argon. N⁶⁰ , N^(G), N^(G1)-Tricarbobenzoxy-L-arginine (77 mg, 0.133 mmol), 1-3-(dimethylamino)propyl!-3-ethylcarbodiimide HCl (25 mg, 0.13 mmol) anda crystal of 1-hydroxybenzotriazole were added under argon at roomtemperature. The reaction was stirred for 1 hr, stored overnight in thefreezer, partitioned between ethyl acetate and water, and the organiclayer was washed successively with dilute brine and dilute sodiumbicarbonate solutions. The solvent was evaporated to leave enol ether 4as white crystals (89.5 mg, 94%).

¹ H NMR (CDCl₃, ppm): 1.6-2.0 (16H, m); 2.59 (1H, br s); 3.22 (1H, brs); 3.24 (3H, s); 3.88 (1H, m); 4.03 (1H, m); 4.45 (1H, m); 4.90 (1H, d,J=12 Hz); 5.07-5.21 (6H, m); 6.26 (1H, d, J-7.65 Hz); 7.02 (1H, d,J=7.24 Hz); 7.13-7.37 (18H, m); 8.42 (1H, br s); 9.31 (1H, br s); 9.45(1H, br s).

3-Methoxytricyclo 3.3.1,1³,7 !dec-2-ylidenemethyl)-1-anilinyl-L-Arg (5).Dissolve tri-N-Cbz-Arg enol ether 4 in 2.5 ml absolute EtOH, add 5% Pd/C(106 mg) and 1,4-cyclohexadiene (250 μl) and stir in a pressure tube at70° C. for 2 h. Filter the solution through a celite plug, rinse theplug well with water and evaporate the solution to give the deprotectedarginine enol ether 5. This intermediate is used to synthesize arginineenol ether derivatives 6-8 as described below.

3-Methoxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)-1-anilinyl-Arg-Ala-N-Cbz (6). Arginine enol ether5 is dissolved in anhydrous dimethylformamide: pyridine (1:1) underargon. N-Carbobenzoxyalanine (1.1 eq), 1- 3-(dimethylamino)propyl!-3-ethylcarbodiimide HCl (1.2 eq), and a crystal of1-hydroxybenzotriazole are added under argon at room temperature. Thereaction is stirred until coupling is complete, partitioned betweenethyl acetate and water, and the organic layer is washed successivelywith dilute brine and dilute sodium bicarbonate solutions. The solventis evaporated to give N-Cbz-Ala-Arg enol ether 6. Alternatively,coupling can be effected by a mixed anhydride method using analkylchloroformate such as isobutylchloroformate in the presence ofN-methylmorpholine (Smith and Bissell, 1981, U.S. Pat. No. 4,294,923).

3-Methoxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)-1-anilinyl-Arg-Gly-Gly-N-CBz (7).N-Cbz-Gly-Gly-Arg enol ether 7 is synthesized from enol ether 5 asdescribed for compound 6, where N-carbobenzoxyglycine-glycine is used.

3-Methoxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)-1-anilinyl-Arg-Gly-Val-N-Cbz (8).N-Cbz-Val-Gly-Arg enol ether 8 is synthesized from enol ether 5 asdescribed for compound 6, where N-carbobenzoxyvaline-glycine is used.

3-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-L-Arg (9). A solution of arginine enol ether 5in 1:1 CHCl₃ :acetone and 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP,1.5 ml of a 2% solution in CHCl₃ by weight) is cooled to -78° C. in adry ice/acetone bath and irradiated with a 250W, high pressure sodiumlamp while passing a stream of oxygen through the solution. A 5-milpiece of Kapton polyimide film (DuPont) placed between the lamp and thereaction mixture filters out unwanted UV radiation. Upon completion ofthe photooxygenation, the solution is warmed to room temeperature andthe solvent is removed under vacuum to yield arginine dioxetane 9.

3-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-L-Arg-Ala-N-Cbz (10). N-Cbz-Ala-Arg dioxetane10 is synthesized from enol ether 6 as described for compound 9.

3-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-L-Arg-Gly-Gly-N-Cbz (11).N-Cbz-Gly-Gly-Arg-dioxetane 11 is synthesized from enol ether 7 asdescribed for compound 9.

3-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-L-Arg-Gly-Val-N-Cbz (12).N-Cbz-Val-Gly-Arg-dioxetane 12 is synthesized from enol ether 8 asdescribed for compound 9.

3-Methoxytricyclo 3.3.1.1³,7!dec-2-ylidenemethyl)-1-anilinyl-Ala-Ala-N-Cbz (13). Enol ether aniline1 (68 mg, 0.25 mmol) was dissolved in 0.6 ml anhydrous dimethylformamideand 0.6 ml anhydrous pyridine under argon. N-Cbz-Ala-Ala (92 mg, 0.31mmol), 1- 3-(dimethylamino)propyl!-3-ethylcarbodiimide HCl (59 mg, 0.31mmol) and a crystal of 1-hydroxybenzotriazole were added under argon atroom temperature. The reaction was stirred for 2 hr, partitioned betweenethyl acetate and water, and the organic layer was washed successivelywith dilute brine and dilute sodium bicarbonate solutions. The organiclayer was dried over Na₂ SO₄, evaporated and purified on a silica gelcolumn (25-75% EtOAc/hexanes) to yield 134 mg (97%) of enol etherpeptide 13.

¹ H NMR (CDCl₃, ppm): 1.35-1.42 (6H, m); 1.5-2.3 (12H, m); 2.61 (1H, brs); 3.21 (1H, br s); 3.26 (3H, s); 4.15-4.32 (1H, m); 4.62-4.70 (1H, m);5.09 (2H, s) ; 5.5 (1H, br s); 6.8 (1H, br s); 7.02 (1H, d, J=7.6 Hz);7.30 (5H, m); 7.2-8.0 (4H, m).

3-Methoxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)-1-anilinyl-Ala-Ala(14). Enol ether 13 (28.7 mg, 0.05 mmol) was dissolved in 1 ml absoluteEtOH.

Palladium on carbon catalyst (5%, 33 mg) and 1,4-cyclohexadiene (40 μl,0.5 mmol) were added and the reaction was heated stirring at 55° C. for40 min. The solution was filtered through a celite plug to removecatalyst and evaporated to yield 18 mg of 14 as clear oil. The crude oilwas used without further purification for subsequent peptide couplings.

3-Methoxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)-1-anilinyl-(Ala)₃-N-Ac (15). N-Ac-(Ala)₃ enol ether 15 is synthesized by coupling (Ala)₂-enol ether 14 with N-acetyl-L-Ala as described for compound 13.

3-Methoxytricyclo 3.3.1.1³,7 !dec-2-ylidenemethyl)-1-anilinyl-(Ala)₄-N-Cbz (16). N-Cbz-(Ala)₄ -enol ether 16 was synthesized by coupling(Ala)₂ -enol ether 14 (18 mg, 0.04 mmol) with Ala-Ala-N-Cbz (14 mg, 0.48mmol) as described for compound 13.

3-(4-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-Ala-Ala-N-Cbz (17). N-Cbz-Ala-Ala-dioxetane 17is synthesized from enol ether 13 as described for dioxetane 3.

3-(4-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-(Ala)₃ -N-Ac (18). N-Ac-(Ala)₃ -dioxetane 18 issynthesized from enol ether 15 as described for dioxetane 3.

3-(4-Methoxyspiro 1,2-dioxetane-3,2'tricyclo 3.3.1.1³,7!decan!-4-yl)-1-anilinyl-(Ala)₄ -N-Cbz (19). N-Cbz-(Ala)₄ -dioxetane 19is synthesized from enol ether 16 as described for dioxetane 3.

    ______________________________________    Scheme 1.    Synthesis of Protease Dioxetane Substrates    ______________________________________     ##STR3##     ##STR4##    Enol Ether Peptide                    Where the Peptide is:    ______________________________________    2               LeuGlyGlyNCbz    4               Arg-tri-NCbz    5               Arg    6               ArgAlaNCbz    7               ArgGlyGlyNCbz    8               ArgGlyValNCbz    13              AlaAlaNCbz    14              AlaAla    15              (Ala).sub.3 NAc    16              (Ala).sub.4 NCbz    ______________________________________     ##STR5##     ##STR6##    Dioxetane Peptide              From Enol Ether                           Where the Peptide is:    ______________________________________     3        2            LeuGlyGlyNCbz     9        5            Arg    10        6            ArgAlaNCbz    11        7            ArgGlyGlyNCbz    12        8            ArgGlyValNCbz    17        13           AlaAlaNCbz    18        15           (Ala).sub.3 NAc    19        16           (Ala).sub.4 NCbz    ______________________________________     Where X, Y = H, Cl, Br, l, F OMe, OH, OR, alkyl, COOR, CONHR, CONR2, etc.     Where Cbz = NCarbobenzoxy and Ac = Nacetyl

CHEMILUMINESCENT ASSAY FOR SUBTILISIN

Subtilisin is a thermally stable enzyme capable of withstanding 60° C.temperatures. It is also tolerant of salts and resistant tourea/detergent denaturation.

Protocol

Experiment 1

Protease substrate 3 (0.1 mM in 0.05M Tris, pH 8.5, 10% acetonitrile)was incubated at 30° C. in a Turner TD-20e Luminometer. After recordingthe background emission for approximately 4.5 minutes, l, of 20 mg/mLprotease (Subtilisin Carlsberg, Sigma P5380) was added and the lightemission was measured. The background was measured on the 0-10 scale.Upon addition of enzyme, the scale was changed to the 0-100 scale andlater (around 9 minutes) to 0-1000. The results of this experiment areshown in FIG. 1.

Experiment 2

Protease (Subtilisin Carlsberg Sigma P5380) was serially diluted andadded to 0.1 mM protease substrate 3 in 0.05M Tris, pH 8.5 containing10% acetonitrile, 1 mg/mL Sapphire, or 0.1% Pluronic F127. The followingconcentrations of protease were tested in duplicates (100 μL per well):200, 20, 2, 0.2, and 0.02 μg per mL. The microtiter wells were thenincubated at 37° C. for 1 hour and the chemiluminescent signal wasmeasured in a Dynatech ML2250 Microtiter plate luminometer. The platewas then incubated for another hour at 45° C., and the chemiluminescentsignal was again measured. The results of this experiment are shown inFIG. 2 (37° C.) and FIG. 3 (37° C. followed by 45° C.).

As shown by the above experiment, digestion of the peptide moiety Z bythe enzyme results in spontaneous decomposition and chemiluminescence.One particular application, by no means intended to be limiting to theinvention, embraces DNA detection in solution, and DNA blot assays,using thermophilic enzymes such as thermolysin. The preparation of DNAmaterials frequently requires high temperature processes, includingdenaturation and PCR amplification. It would be advantageous to affixthe enzyme label to the DNA in advance of these high temperatureprocesses, and have the label carried forward. Many enzymes, however,are deactivated and denatured by exposure to high temperatures, andaccordingly, complex post-amplification coupling requirements arefrequently used. As the novel dioxetanes of the claimed inventionfunction as excellent substrates for thermolysin and other thermophilicenzymes, this invention finds particular application to DNA detection,and DNA hybridization assays. In particular, DNA probes and DNA samplescan be prepared with the thermophilic enzyme affixed, subjected todenaturation and amplification, subsequently prepared in solution ordeposited on a membrane, subjected to stringent hybridizationconditions, and still retain the enzyme label which will activate thedioxetane substrate reporter molecule of the invention.

Other assay formats familiar to those of skill in the art can beemployed in connection with the claimed invention. In addition tosingle, solution phase assays for the presence of a particular enzyme,labels can be attached via covalent bonding, to special or high-affinitybonding ligands, such as antibodies, antigens and ligand pairs such asavidin or stretavidin-biotin bonding pairs. Thus, in addition to DNAhybridization assays, DNA solution assays, enzyme assays, otherconventional assays including immunoassays and specific solution assayscan be practiced within the claimed invention.

Additionally, as illustrated in FIGS. 2 and 3, a wide variety ofenhancement agents can be used to improve chemiluminescent performance.Representative enhancement agents, such as those set forth in U.S. Pat.No. 4,978,614 are typically water-soluble macromolecules, which may benaturally occurring substances, such as albumin, or a polymericquaternary onium salt, including phosphonium, sulfonium and ammoniumsalts. Exemplary enhancement agents are disclosed in U.S. Pat. Nos.5,145,772, and 5,112,960, includingpoly(vinylbenzyltrimethylammonium-chloride) (TMQ),poly(vinylbenzyltributylammoniumchloride) (TBQ) andpoly(vinylbenzyldimethylbenzylammoniumchloride) (BDMQ). These particularpolymers act to sequester the hydrophobic dioxetane amine, excludingwater, which tends to dampen or "quench" dioxetane chemiluminescence.Further improvements can be obtained by using an enhancement additivewhich further improves the ability of these enhancement agents to formhydrophobic regions, particularly surfactants, negatively charged salts,alcohols, turpentines and other solvents, and water-soluble polymers.Typical enhancement effects obtained through this method are disclosedin U.S. Pat. No. 5,547,836, which is incorporated herein by reference.Where a blotting assay, or other membrane-based assay is to bepracticed, chemiluminescent performance, and S/N ratio, as well assensitivity, can be further enhanced by using membranes coated with apolymer coating, including those disclosed in U.S. Pat. No. 5,336,596,which is incorporated herein by reference. Suitable supports for thepolymer coating include nitrocellulose, nylon and PVDF.

As noted above, the dioxetane amine remaining after removal of theprotease-labile moiety is akin to the oxyanion of aryloxy-substituteddioxetanes of the prior art, in that it spontaneously decomposes andchemiluminesces. The dioxetane amine-based assay may be improved bygentle heating of the solution or sample comprising the dioxetane amine.The dioxetanes of this invention are particularly stable, andaccordingly, gentle heating should not give rise to substantialincreases in the spontaneous emission of light caused by thermallyinduced decomposition. Other elements of the sample, that is, theelements of the sample being inspected, including the target analyte,will generally establish the limits as to what range of elevatedtemperatures may be employed. The dioxetanes and assays of thisinvention employ proteases, which are generally subject to denaturingand/or activity reduction at elevated temperatures (although some areresistant to most conventional temperatures) and frequently findimportant applications in the inspection of biological samples anddetection of analytes which may additionally be heat sensitive. Agenerally recognized temperature limit for biological material is 100°F. Accordingly, where the assay employs biological materials, heatingabove 100° F. should be avoided. A generally preferred range is 45°-65°F. Heating at temperatures between ambient temperatures and 45° F. mayrealize a more modest increase in reaction speed.

This invention has been disclosed in terms of both generic descriptionand specific example. Variations will occur to those of ordinary skillin the art, including peptide moiety identities, specific proteolyticenzymes to be employed, enhancement agents and enhancement additives,and specific assay formats without the exercise of inventive faculty.Such variations remain within the scope of the invention, save forvariations excluded by the recitation of the claims presented below.

What is claimed is:
 1. A compound of the formula: ##STR7## wherein X¹,X² and Y are independently hydrogen or an electron donating or electronwithdrawing substituent, wherein R is an alkyl, straight-chain orbranched chain of 1-20 carbon atoms or cycloalkyl or polycycloalkyl of3-20 carbon atoms, and wherein Z is an amino acid, or a polypeptide,wherein Z is an enzyme-specific substrate andwherein an enzyme specificfor said group Z is selected from the group consisting of AmyloidA-4-generating enzymes, Aminopeptidase, Cathepsin B, Cathepsin C,Cathepsin D, Cathepsin G, Cathepsin H, Cathepsin L, Elastase,Endopeptidase, Subtilisin, Thermolysin, tPA and Urokinase.
 2. Thecompound of claim 1, wherein at least one of X¹, X² and Y is an electrondonating or electron withdrawing group, and is further selected so as tobe a solubilizing group for rendering said compound more soluble inaqueous preparations.
 3. The compound of claim 1, wherein X¹ and X² are,individually hydrogen, a hydroxyl, a halogen, a hydroxy lower alkylgroup, a halo lower alkyl group, a phenyl group, a halo phenyl group, analkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amidegroup.
 4. The compound of claim 1, wherein Y is hydrogen, chloro,alkoxy, aryloxy, trialkylammonium, alkylamido, arylamido, arylcarbamoyl,alkylcarbazoyl, cyano, nitro, ester, alkyl- or arylsulfonamido,trifluoromethyl, aryl, alkyl, trialkyl-, triaryl, or alkylarylsilyl,alkyl- or arylamidosulfonyl, alkyl- or arylsulfonyl and alkyl- orarylthioether.
 5. The compound of claim 2, wherein said solubilizinggroup is selected from the group consisting of an ammonium group, aphosphonium group, a sulfonium group, a carboxylic acid group, asulfonic acid group, a trifluoromethylsulfonyl group, a methylsulfonylgroup, a cyano group and a hydroxy group.
 6. The compound of claim 1,wherein at least one of X¹ and X² is chloro, and Y is chloro or methyl.7. The compound of claim 1, wherein A is ##STR8##
 8. A method ofdetecting the presence and/or amount of a protease in a sample,comprising adding the compound of claim 1 to said sample, incubatingsaid sample and inspecting said sample for the generation of light,wherein light so generated is indicative of the presence and/or amountof said protease, wherein Z of said compound is an amino acid orpolypeptide substrate for which said protease is specific.
 9. The methodof claim 8, wherein said protease is bound to DNA.
 10. The method ofclaim 8, wherein said protease is bound to an antibody.
 11. The methodof claim 8, wherein said protease is bound to a substance for which anantibody is specific.
 12. The method of claim 8, wherein said proteaseis bound to avidinor streptavidin-biotin bonding pairs.
 13. A kit forthe detection of a protease by generation of chemiluminescence,comprising a compound of claim 1 and a membrane on which an assay forsaid detection of a protease employing said compound is conducted.
 14. Akit for conducting an assay to determine the presence and/or amount of aprotease in a sample comprising of a compound of claim 1, and asubstance which enhances the amount of light released by the removal ofmoiety Z from said compound, as compared with the amount of lightgenerated by the removal of moiety Z in the absence of said enhancer.15. The kit of claim 13, wherein said kit further comprises a membraneon which said assay is conducted.